non-invasive biomarker signatures as a new tool for stratifying...
Post on 01-Jan-2021
1 Views
Preview:
TRANSCRIPT
Non-invasive biomarker signatures as a new tool for stratifying
biopsy indications?
Blood B cell profile as Biomarker in kidney transplantation?
Sophie BrouardSophie Brouard
ITUN, INSERM U643-ITERT, Nantes, France
“The exposure of mice to foreign antigens at a foetal stage induced tolerance to a skin graft expressing the same antigens at the adult stage”
Well fonctioning graftLacking histological lesion of rejectionIn the absence of immunosuppressionIn an immunocompetent hostAccepting a second graft of the same donorWhile able to reject a third party graft
LimitsLimits in in humanhuman tolerancetolerance??
=> Several key elements of transplant tolerance in rodents cannot be demonstrated…• « clinical state » observed « by chance » and not in the context of planned tolerance
inducing protocols• Patients usually incompliant• Patients usually incompliant• Graft biopsies are often unavailable• Refuse investigative procedures
⇒Functional definition
“Well-functioning graft in the absence of exogenous immunosuppression in an
Immunocompetent host”
OperationallyOperationally toleranttolerant patientspatientsN=2710+17 new casescreatinemia < 150 µmol/Lproteinuria < 1g/dayabsence of immunosuppressive drugs for at least one year
400
DF-Tol Sta CR0
100
200
300
crea
tinem
ia (
µmol
/l)
Identification of blood biomarkers of operational Identification of blood biomarkers of operational tolerance: help for biopsy and clinical decision tolerance: help for biopsy and clinical decision
BenchClinic
High risk Low risk
ToleranceChronic rejection
A challenge for the future:To identify allograft recipients in whom IS withdrawal could be safely
conducted
Transcriptional profiling of blood samples from operationally
tolerant transplant recipients
published experiences
� Martínez-Llordella et al. Am J Transplant 2007 (16 tolerant patients)
Liver
� Brouard et al. PNAS 2007 (17 tolerant patients)
� Sagoo et al. J Clin Invest 2010 (11 tolerant patients)
� Newell et al. J Clin Invest 2010 (25 tolerant patients)
Kidney
� Martínez-Llordella et al. Am J Transplant 2007 (16 tolerant patients)
� Martínez-Llordella et al. J Clin Invest 2008 (28 tolerant patients)
Transcriptional profiling of blood samples from operationally
tolerant kidney transplant recipients17 tolerant recipients
8 healthy individuals
22 chronic rejection
10 minimally immunosuppressed
12 immunosuppressed stable kidney recipients
14 acute rejection Healthy individuals
Chronic
Training set
5 8Tolerantrecipients
Testing set12 Tolerant11 Chronic rejection
49-gene predictivesignature
Correct classification in 22/23
Chronicrejection
11
BrouardBrouard et alet al. . ProcProc NatlNatl AcadAcad SciSci 20072007
Gene Definition Fold TOL vs. CR Fold TOL vs. N
TK1* Thymidine kinase 1, soluble 6.36 6.95
DHRS2* Dehydrogenase/reductase (SDR family) member 2 5.63 7.48
CCNB2 Cyclin B2 5.10 3.91
CYR61* Cysteine-rich, angiogenic inducer, 61 5.08 4.55
AFP* Alpha-fetoprotein 4.86 6.52
COL3A1 Collagen, type III, alpha 1 3.76 4.28
STK6* Serine/threonine kinase 6 3.59 5.27
SERPINA3* Alpha-1 antiproteinase, antitrypsin member 3 3.43 5.20
NGFRAP1 TNFRSF16 associated protein 1 3.20 2.06
IFITM3 Interferon induced transmembrane protein 3 (1-8U) 3.16 2.10
CTGF Connective tissue growth factor 3.07 4.66
CCNB1 Cyclin B1 2.63 3.14
LTBP4 Latent TGF beta binding protein 4 2.62 2.04
SOX3* SRY (sex determining region Y)-box 3 2.50 4.18
8
SOX3* SRY (sex determining region Y)-box 3 2.50 4.18
MTB More than blood homolog 2.44 2.03
KIFC3 Kinesin family member C3 2.29 3.20
NKG7 Natural killer cell group 7 sequence 1.93 0.84
TCL1A T-cell leukemia/lymphoma 1A 1.80 0.52
CD79A CD79A antigen (Ig-associated alpha) 1.66 0.70
CD79B CD79B antigen (immunoglobulin-associated beta) 1.41 1.03
RPS5 Ribosomal protein S5 1.38 2.18
CD22 CD22 antigen 1.15 0.41
CD19 CD19 antigen 0.79 0.37
CD20 Membrane-spanning 4A1 (MS4A1) 0.73 0.13
BCNP1 B-cell novel protein 1 0.69 0.34
DPYD Dihydropyrimidine dehydrogenase 0.25 0.46
EGR1 Early growth response 1 0.15 0.42
EVI2A Ecotropic viral integration site 2A 0.11 0.46
Pangenomic microarray (Brouard et al. PNAS 2007)
Enrichement of genes related to B cell pathways (GSEA)Biological function analysis (Gominer)
7 major gene sets related to B cell pathways (Pallier et al. Kidney Int. 2010)
Z= 15
Gene set nameNumber of genes
NESNOM p-val
FDR q-val
Gene set description References
BASSO_REGULATORY_HUBS
61 1.80 0.006 0.21Genes highly interconnected in the reconstructed regulatory networks from expression profiles in human B cells.
Basso K et al., Nat Genet. 2005
HOFFMANN_BIVSBII_BI_TABLE2
100 1.73 0.005 0.19Genes with at least five fold change in expression between Pre-BI and Large Pre-BII cells.
Hoffmann R et al., Genome Res. 2002
TARTE_PLASMA_BLASTIC
160 1.71 0.014 0.20
Genes overexpressed in mature plasma cells isolated from tonsils and mature plasma cells isolated from bone marrow as compared to polyclonal plasmablastic cells.
Tarte K et al., Blood 2003
ZHAN_MM_CD138_PR_VS_REST
22 1.70 0.014 0.21
50 top ranked over-expressed genes in each of the seven subgroups of multiple myeloma in CD138-enriched plasma cells from 414 newly diagnosed
Zhan F et al., Blood 2006S_REST enriched plasma cells from 414 newly diagnosed
patients.Blood 2006
GREENBAUM_E2A_UP 15 1.54 0.014 0.24Genes up-regulated at least 3-fold in the E2A-deficient cell lines.
Greenbaum S et al., J Biol Chem. 2004
HOFFMANN_BIVSBII_LGBII
60 1.53 0.013 0.24Genes with at least five fold change in expression between large and small Pre-BII cells.
Hoffmann R et al., Genome Res. 2002
TARTE_PC 38 1.59 0.015 0.25
Genes overexpressed in polyclonal plasmablastic cells, mature plasma cells isolated from tonsils and mature plasma cells isolated from bone marrow as compared to B cells purified from peripheral blood and tonsils.
Tarte K et al., Blood 2003
⇒ Several B cell-associated subsets with up-regulation of genes related to cell cycle, proliferation, development and maturation. (Pallier et al. Kidney int 2010)
Tolerant patients are characterized by a higher number of peripheral B cells
B c
ells
Louis et al. Transplantation 2006
B c
ells
B cells modulate T cell immunity
•The presence of infiltration of B cell compartments is actua lly correlated withworse prognosis
•Antigen presentation by B cells•B cell cytokines•B cell costimulation of T cells•B cells form ectopic lymphoid organs•Antibody mediated immune enhancement
•B cells and regulation of alloimmune response?12
CR DF STA HV0.0
2.5
5.0
7.5
10.0
12.5*
*
CD
19+ B
cel
ls(F
requ
ency
)
CR DF STA HV0
50
100
150
200
250
300
350 * **
*
**
CD
19+ B
cel
ls/µ
l
Operationally tolerant patients display a higher number of memory B cellsPallier et al. Kidney Int. 2010
***
CR DF STA HV0
10
20
30
40
50
60
70
80
****
****
IgD
CD
38+
/- B
cel
ls/µ
l
CR DF STA HV0
10
20
30
40
50
60
70
80**
**
***
***
CD
19+ C
D27
+ B c
ells
/µl
0
1
2
3** ***
*
IgD
- CD
38+
/- C
D80
+ B c
ells
/µl
0.0
0.5
1.0
1.5
2.0 ** ***
IgD
- CD
38+
/- C
D86
+ B c
ells
/µl
DF patients display a high number of circulating B cells that express a memory phenotype and over-express costimulatory molecules.
CR DF STA HV0Ig
D
CR DF STA HV0.0Ig
D
Pallier et al. Kidney Int. 2010
CR Tol STA HV0
50
100*
IgD
- CD
38+
/- CD
40+ B
cel
ls/µ
l
CR DF STA0
1000
2000
3000
4000
5000
6000* **
CD
40/I
gD- C
D38
+/- E
BM
5 B
M5
mea
n flu
ores
cenc
e in
tens
ity
Sagoo et al. J. Clinical. Invest. 2010Newell et al. J. Clinical. Invest. 2010
CD79b
TCL1A
Rank120
5
10
15
***
units
/HP
RT
1.5
2.0 *
units
/HP
RT
1.0
1.5
2.0 *
units
/HP
RT
*****
Tolerant patients exhibited a distinct pattern of gene expression
•Significant enrichment of several B-lymphocyterelated pathways
•6/10 of the genes with most altered levels ofexpression areexpressed in B cells
BLKHS3ST1
SH2D1BTCL1A
Rank 329Rank 1
Rank 3 Rank 111
0.0
0.5
1.0
1.5
units
/HP
RT
0.0
0.5
1.0
units
/HP
RT
0.00
0.02
0.04
0.06
0.08
0.10 **
units
/HP
RT
0
1
2
3
**
units
/HP
RT
******
*****
Sagoo et al. J. Clinical. Invest. 2010
Newell et al. J. Clinical. Invest. 2010
•Blood B cell signature: Immunoglobulin variable region genes
•Urine B cell signature
524 6 433
Lozano et al.
[808]
Sagoo et al.
[946]
35
3971
ADAM28 *AFF3 *ASB2ASPMBACE2BTLA *CD19 *CD200 *
ID3 *IGHM *IGKC *IGLL3 *KRT18MGC29506 *MS4A1 *PEX3
Newell et al.
[4721]
CD200 *CD22 *CD79A *CNOT2DTX1 *EBF1 *FCER2 *FCRL2 *FNBP4 *GNG7 *HLA-DOB *
PEX3PLEKHG1 *PNOC *QRSL1 *RFC1RFC4RPS21SWAP70 *ZNF264ZNF606
* B cell genesLozano et al. Am. J. Transplant. In press
DiLillo et al. Ann. N. Y. Acad.Sci. 2010
CR DF STA HV0
5
10
15
20
25 *
CD
19+ C
D5+
B c
ells
(Fre
quen
cy)
Pallier et al. Kidney Int. 2010
CD
24
CD38
Memory CD27 +IgD -
Naïve/Mature
Transitional
Newell et al. J. Clinical. Invest. 2010
10
20 *
(CD
32a/
CD
32b)
mR
NA
/HP
RT
0.4
0.6
0.8
**
CD
32b
mR
NA
/HP
RT
*
B cells from operationally tolerant recipients display an inhibitory profile
DF-patients had a significantly decreased CD32a/CD32b ratio compared to patients with chronic rejection (p<0.05) in total blood.
CR DF STA HV0(C
D32
a/C
D32
b)m
RN
A/H
PR
T
CR DF STA HV0.0
0.2
CD
32b
mR
NA
/HP
RT
FcγγγγRIIA (CD32a) transduces an activator signalFcγγγγRIIB (CD32b) contains an immunoreceptor tyrosine-based inhibition motif, transducing an inhibitory signal upon co-ligation with the B cell receptor.The CD32a/CD32b ratio has been shown to be a relevant index of B cell activation/inhibition(Ravetch et al. Science, 2000; Ono et al. Nature, 1996)
Pallier et al. Kidney Int. 2010
Key leader gene
2
3
4* **
BA
NK
1 m
RN
A/H
PR
T
B cells from operationally tolerant recipients display an inhibitory profile
CR DF STA HV0
1
BA
NK
1 m
RN
A/H
PR
T
Modulator of hyperactive B cell responses by inhibi ting AKT activation upon CD40 signalling (Aiba et al. Immunity, 2006)
Pallier et al. Kidney Int. 2010
Sivozhelezov V. et al.JCB, 2008
FcγγγγR2b PBMC
Le Texier et al. Am. J. Transplant. 2010
What about B cell Transcriptional profiling of blood samples
from operationally tolerant liver recipients?
� Brouard et al. PNAS 2007
Kidney
Liver
6.7%
33.3%
60%
20
30
40
50
60
70
%
3-6 years
6-10 years
over 10
years
� Martínez-Llordella et al. Am J Transplant 2007
� Martínez-Llordella et al. J Clin Invest 2008
6.7%
0
10
A. Sanchez, Barcelona, personal communication
DF STA0
5
10
15*
CD
19+ B
-cel
ls (
Fre
quen
cy)
Patients and MethodsSTUDY SUBJECTS:
Liver-TOL (n=12): HCV-negative liver recipients off IS for >1 year maintaining stable
graft function liver histology.
Liver-NonTOL (n=12): HCV-negative liver recipients maintaining stable graft function
under maintenance IS but who underwent rejection during a previous weaning
attempt.
Kidney-TOL (n=12): kidney recipients without IS with stable graft function for at
least 3 years (serum creat <150µmol/L and proteinuria<1g/24h).
Kidney-STA (n=12): kidney recipients under maintenance IS with stable graft
function for at least 3 years (serum creat <150µmol/L and proteinuria<1g/24h).
Kidney-CR (n=12): kidney recipients with deteriorating graft function (serum creat
>150µmol/L and/or proteinuria>1g/24h) with either transplant glomerulopathy or C4d
deposits plus DSA.
HV (n=12): healthy individuals
METHODS:
� PBMC samples
� Gene expression analyses: Affymetrix U133 Plus 2.0 microarrays
Different PBMC subsets contribute to the kidney and liver tolerance-related expression patterns
LIVER
Z=3.685
all genesCD56-specific genes
all genesCD19-specific genes
Z=-22
Den
sity
CD56 CD19
Haematology Expression Atlas Gene Sets(Watkins NA et al. Blood 2009): CD14, CD56, CD4, CD8, CD19, CD16
KIDNEY
all genesCD19-specific genes
Z= 15
all genesCD56-specific genes
Z= -9
all genesCD14-specific genes
Z= -21
CD56CD19 CD14
Absence of overlap between the kidney and liver tolerance -related datasets
UPDOWN
2610
20
621372
2610
10
35472
Liver-TOLvs
Liver-NonTOL
Kidney-TOLvs
Kidney-Sta
DOWN0 0 372
FDR<10%
Liver-TOLvs
Liver-NonTOL
Kidney-TOLvs
Kidney-CR
0 0 472
Liver Kidney Liver Kidney
28
Liver Kidney
Liver Kidney
CD
19+C
D5+
B c
ells
Lordano et al, AJT In press
Conclusions
•B cells and B cell-related transcripts are enriched in blood from tolerant
kidney recipients.
•This is a highly reproducible observation common to all cohorts of kidney
tolerant recipients.
•There are no obvious similarities for B cells between blood from kidney and•There are no obvious similarities for B cells between blood from kidney and
liver tolerant recipients (at least similarities that are reproducible across all
studies).
•This analysis is restricted to peripheral blood and has a number of limitations
����B cells: markers or key players?
•Large studies
•Access to the biopsy…
Operationally tolerant Kidney recipients: An heter ogenous group…
Complex and multifaceted clinical concept with poss ibly truly tolerant recipients
Some patients keep a functional graft for decades ( >30years)No opportunistic infection (Roussey et al. Am. J. Tr ansplant 2005)Normal immune response (Ballet et al. Transplantati on 2007)17 patients without/ 8 patients with HLA Abs (4 wit h DSA)
Metastable in some patients
9/27 decline their functionTo what extend the rupture of the operational toler ance is driven by To what extend the rupture of the operational toler ance is driven by
immunological phenomena is not clear yet….
Histology C4d Banff scores DSA
Case 4 Transplant Glomerulopathy -g2, i0, t0, v0, ptc1,
cg2, mm2, ti2, ci3, ct3, cv1, ah3 ND
Case 5 Borderline -g0, i1, t1, v0, ptc0,
cg0, mm0, ti1, ci1, ct1, cv2, ah3 -
Case 7 Transplant Glomerulopathy -g2, i0, t0, v0, ptc0,
cg2, mm2, ti1, ci2, ct2, cv1, ah1 +
Case 9 IF/TA grade 1 -g0, i0, t0, v0, ptc0,
cg0, mm0, ti0, ci1, ct1, cv2, ah1 -
Case 10Transplant Glomerulopathy
and Chronic active TCMR
-g3, i0, t0, v0, ptc1,
cg3, mm2, ti3, ci3, ct3, cv3, ah3 -
Case 13 IF/TA grade 3 NDgx, i1, t0, v0, ptc3,
cgx, mmx, ti2, ci3, ct3, cvx, ah0 -
“Acceptation” rather than “tolerance”?
« Lack of a destructive immune response toward the graf t »
“Old” kidneys may actually be tolerated despite a slo w and physiological loss of function over years…
AknowledgementsInserm U643
Annaick PallierSophie Hillion
Richard DangerKarine Renaudin
Cécile Guillot-GuegenSandrine LerouxYohann FoucherNicolas Degauque
ITUN-INSERM U643 (France)
Nicolas DegauqueAnne Devys
Anne Cesbron-Gauthier
Jean-Paul Soulillou/Magali Giral
Coll. A. SanchezColl. A. Sanchez--FueyoFueyo, , BarceloneBarcelone, Spain, Spain
G. Roussey, J-F. Subra, F. Villemain, C. Legendre, F.J. Bemelman, G. Orlando, A. Garnier, H. Jambon, H. Le Monies De Sagazan, L. Br aun, C. Noël, E.
Pillebout, M-C. Moal, C. Cantarell, A. Hoitsma, M. Ranbant, A. Testa
top related